The Roles of Gut Microbiota Metabolites in the Occurrence and Development of Colorectal Cancer: Multiple Insights for Potential Clinical Applications

Colorectal cancer (CRC) is one of the most common cancers worldwide. The occurrence and development of CRC are related to multiple risk factors such as gut microbiota. Indeed, gut microbiota plays an important role in the different phases of colorectal cancers (CRCs) from oncogenesis to metastasis. Some specific bacteria such as Fusobacterium nucleatum (F. nucleatum) associated with CRCs have been found. However, recently identified bile acid and tryptophan metabolites as well as short chain fatty acids (SCFAs), which are derived from gut microbiota, can also exert effects on the CRCs such as that SCFAs directly inhibit CRC growth. Importantly these metabolites also modulate immune responses to affect CRCs. They not only act as tumor inhibiting factor(s) but also promotor(s) in the occurrence, development, and metastasis of CRCs. While gut microbiota metabolites (GMMs) inhibit immunity against CRCs, some of them also improve immune responses to CRCs. Notably, GMMs also potentially affect the shaping of immune-privileged metastatic niches through direct roles or immune cells such as macrophages and myeloid-derived suppressive cells. These findings offer new insights for clinical application of gut microbiota in precise and personalized treatments of CRCs. Here, we will mainly discuss direct and indirect (via immune cells) effects of GMMs, especially SCFAs, bile acid and tryptophan metabolites on the occurrence, development and metastasis of CRCs.


Introduction
C olorectal cancer (CRC) is one of the most common cancers and accounts for approximately 10% of all annually diagnosed cancers. 1,2The occurrence and development of CRC are related to multiple risk factors such as gut microbiota.Indeed, gut microbiota not only plays a critical role in normal gut physiological functions such as digestion, biosynthesis of vitamins, generation of heat, gut immunity, and maintenance of gut homeostasis, but also contributes to a wide range of diseases such as tumor.
The roles of gut microbiota in the occurrence and development of CRCs have been widely reviewed.Multiple factors of gut microbiota such as gut dysbiosis, specific pathogenic microbes, metabolites and virulence factors can contribute to the initiation and progression of CRCs.Although there exist debates surrounding the causal relationship between microbial dysbiosis, which is compositional and functional alterations of the gut microbiome, 3 and colorectal tumorigenesis, 4 a number of studies have shown that the dysbiosis of gut microbiota is related to CRC development and progression.The characteristic changes in gut microbiota can happen in various CRC stages, including early and advanced CRCs in CRC patients.Recent metagenomic and metabolomic analyses showed distinct gut microbiome-derived phenotypes in early onset of CRCs. 5 There were enriched Flavonifractor plauti and increased tryptophan (Trp), bile acid (BA), and choline metabolism, whereas in late-onset CRC, Fusobacterium nucleatum enrichment, short-chain fatty acid depletion, reduced microbial gamma-aminobutyric acid biosynthesis and a shift in acetate/acetaldehyde metabolism towards acetylcoenzyme A production were found 5 Indeed, specific bacteria associated with the onset and progression of CRCs have been found, such as Fusobacterium nucleatum (F.nucleatum), Enterococcus faecalis (E.faecalis), Streptococcus gallolyticus (S. gallolyticus), Bacteroides fragilis (B.fragilis), and Escherichia coli (E.coli).The metabolites from gut microbiota can not only cause initial inflammation but also play an important role in the CRCs from oncogenesis to metastasis.Mechanistically, CRCs can be derived from the metabolites mediated genetic and epigenetic pathways such as histone modifications, DNA methylation, and noncoding RNAs. 6However, gut microbiota can also produce a wide range of anticancer metabolites, including bacteriocins, antibiotics, peptides, enzymes, and toxins to inhibit tumor growth.
Notably, recent studies have shown that gut microbiota metabolites (GMMs) such as short chain fatty acids (SCFAs), BA, and Trp metabolites not only directly influence tumor growth 7 but also indirectly affect genetic and epigenetic regulation and the metabolism in the immune cells, including myeloid-derived immune cells such as macrophages (Macs) and myeloid-derived suppressive cells (MDSCs), and lymphoid-derived immune cells such as regulatory T cells (Tregs) and regulatory B cells (Bregs).These immune cells can express the receptors of SCFAs, Trp, and BA metabolites. 8heir activation may promote the differentiation and alter the functions of immune cells to suppress immune responses to maintain the homeostasis of gut tissues.Notably, gut microbiota mediated immune cells such as immunosuppressive Macs and MDSCs can also assist metastatic dissemination by creating niches which allow tumor colonization. 9However, some GMMs such as SCFAs, BA, and Trp metabolites can also promote antitumor immune responses.Here, we will mainly discuss the effects of recently identified BA and Trp metabolites as well as SCFAs from gut microbiota on the CRCs or CRCassociated immune cells.These GMMs not only directly inhibit tumor growth but also indirectly promote development of CRCs.Especially, they not only suppress immune responses but also promote immunity against CRCs.

Gut Microbiota and Its Metabolites
SCFAs, BA, and Trp metabolites from gut microbiota not only directly affect CRCs but also indirectly regulate gut and systemic immune response to influence the occurrence, development and metastasis of CRCs.

Inhibition on CRCS by GMMs
Gut microbiota-derived peptides, bacterial toxin, bacteriocins, bacterial enzymes, and antibiotics are directly against cancer.However, recent studies found that SCFAs, BA, and Trp metabolites from gut microbiota can also inhibit CRCs.
exhibited protective actions against colon carcinogenesis.The inhibitory mechanism shows a close association with the inhibition of histone deacetylases (HDACs).The transformation of glycolytic metabolism is a dominant characteristic of carcinoma cells. 43Colorectal cells prefer glucose to butyrate as their preferred energy resource due to Warburg effect pathway.Butyrate not only can enter the nucleus directly to inhibit HDAC but also cause a reduction in short-chain acyl-CoA dehydrogenase levels, which are the primary process in the catalyzation of mitochondrial butyrate oxidation. 44This process reduces the auto-oxidation of butyrate in CRC cells to cause accumulation of butyrate in carcinoma cells, thereby restraining CRC development. 45CFAs also inhibit the Wnt/b-catenin signaling pathway by inhibiting HDAC activity, thereby preventing the development of intestinal tumors in a murine model. 46Butyrate as a HDAC inhibitor also participates in the apoptosis of CRC cells. 47Notably, butyrate can protect from intestinal tumor growth by blocking the activation of calcineurin and nuclear factor of activated T cells C3 in mouse and human. 48nterestingly, sodium butyrate could induce ferroptosis in CRC cells through cluster of differentiation (CD)44/secondary lymphoid tissue chemokine 7A11 signaling pathway. 49Tributyrin alleviated gut microbiota dysbiosis to repair intestinal damage in antibiotic-treated mice. 50Acetate also inhibited CRC cell multiplication and triggered CRC cell apoptosis in a dose-dependent manner.These effects of acetate were mediated via suppression of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/protein kinase B) pathway. 51 metabolites.The UDCA, a secondary BA, which has a chemical structure with DCA but unlike DCA, has been demonstrated to inhibit CRC occurrence.While patients with CRCs take UDCA for a long period of time, these patients are less likely to relapse following the removal of the colorectal adenoma.Clinical metagenomic and metabolomic studies have also revealed links between microbial BA metabolism and inflammatory bowal disease and CRC progression. 52Serum concentrations of BAs, particularly downstream microbial metabolites of CA, were strongly associated with increased risk of CRC among women. 52otably, decreasing concentration of hydrophobic BA and increasing hydrophilicity of the bile pool were involved in the suppression of UDCA on CRCs. 53,54UDCA also protected against malignant progression of CRC through TGR5-yesassociated protein. 55In addition, LCA had antiproliferative action on different cancer cell lines. 56p metabolites.Alteration of fecal Trp metabolism mediated by microbiota is involved in the pathogenesis of CRCs.Indeed, higher levels of plasma Trp were associated with lower CRC risk 57 ; whereas increased serotonin, a metabolite of Trp, was related to a higher risk of CRCs.The Kyn-to-Trp ratio was also positively associated with CRCs. 58upplementation with L. reuteri, which produces Trp metabolites, inhibits SREBP2 expression and tumorigenesis in mice with gut flora disequilibrium.In addition, kynurenic acid, a Trp metabolite, could inhibit proliferation of several cancer cell lines including CRCs. 598-hydroxyquinaldic acid, another Trp metabolite inhibited migration of CRC cell lines colorectal cancer cell line and LS-180 and increased the expression of b-catenin and E-cadherin.The Trp metabolism also was effective in inducing the differentiation of Tregs.
Others.Other GMMs are also associated with tumor growth such as that inosine, a purine metabolite of A. muciniphila and B. pseudolongum is effective in controlling cell growth and tumor evolution. 60Ellagitannins and ellagic acid are dietary polyphenols, which are poorly absorbed but extensively metabolized by the human gut microbiota to produce different urolithins (Uros).Produced UroA and isoUroA can induce apoptosis in colorectal tumor cell line caco-2 cells through arresting in S and G2/M phases of cell cycle.Kasimsetty et al 61 also confirm the time-and dose-dependent anticlonogenic and proliferative activity of UroA in human CRC cells (HT-29) through cell-cycle arrest in the G0/G1 and G2/M stages and apoptosis induction.In addition, F. nucleatum-derived succinic acid also induced tumor resistance to immunotherapy in CRCs.

Promotion on CRCs by GMMs
There is a matter of debate in a causal relationship between gut microbiota and colorectal tumorigenesis.However, studies have shown that the dysbiosis of gut microbiota is related to CRCs.Specific gut bacteria, such as Fusobacterium nucleatum, Escherichia coli, and Bacteroides fragilis could be involved in colorectal carcinogenesis.These bacteria produce metabolites, such as H 2 S, trimethylamine-N-oxide (TMAO), which are likely to promote inflammation and subsequently cancer development. 62Previous reports also showed that bacterial toxins such as toxin from B. fragilis, adhesin A from F. nucleatum and colibactin from E. coli 6,63 can promote the occurrence and development of CRCs.They participate in metabolism of dietary components to produce tumorigenic metabolites, interaction with genetic or epigenetic alterations, induction of inflammation and the recruitment of immunosuppressive cells.Indeed, colibactin could cause the breakage of double-strand DNA, chromosome instability, and cell senescence in eukaryotic cells. 64Notably, the gut microbiota also interacted with certain environmental factors such as a high-fat diet (HFD) 65 and cigarette smoking 66 to promote the development of CRCs.The fat-mediated alterations of the gut microbiota linked BA metabolism to CRC risk and colonic tumorigenesis. 67Cigarette smoke also promoted CRC through modulation of gut microbiota and related metabolites. 66However, recent studies have also found that GMMs such as secondary BAs and Trp metabolites are related to the promotion on CRCs.
BA metabolites.The secondary BAs have exhibited procarcinogenic properties.Paired microbiome and metabolome analyses were associated with BA changes with CRC progression.The plasma levels of 7 conjugated BA metabolites, including glycocholic acid, taurine-conjugated CA, glycodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid (TDCA) were positively correlated with risk of colon cancer.Metabolomic analysis showed increased BA metabolite TDCA in the colon of smokeexposed mice 66 and in the colon of individuals consuming HFDs, which were associated with an increased risk of CRCs.HFDs also promoted colorectal tumorigenesis through modulating gut microbiota and metabolites. 65HFD feeding could increase CRC growth in b2AR-dependent manner. 68nterestingly, germ-free mice transplanted with stools from smoke-exposed mice had increased colonocyte proliferation. 66Cigarette smoke could promote CRC through modulation of gut microbiota and related metabolites. 66The secondary BAs fed mice also increased inflammation and induced CRCs. 69Studies found that hydrophobic BAs can produce reactive oxygen species (ROS) and reactive nitrogen substances, which cause oxidative stress, damage to DNA and proteins, and destruction of the base excision repair pathway.In the carcinogenic methane peroxideinduced tumor model system, DCA raises the rate of CRCs in Kirsten rat sarcoma viral oncogene homolog point mutual mutations.DCA can also enhance the local spatial aggregation of phospholipid acid and induces co-localization between phospholipid acid and epidermal growth factor receptor (EGFR) to promote EGFR dimerization/oligomerization and EGFR-MARK signaling.EGFR, as a tyrosine kinase receptor can promote proliferation, invasion or metastasis of CRCs by mutation or overexpression.Notably, unconjugated BAs and tertiary BAs are not associated with cancer risk.
Trp metabolites.Trp metabolites serotonin (5-HT) and 3-hydroxyanthranilic acid (3-HAA) can facilitate tumor cells such as CRCs to escape from ferroptosis.Mechanistically, both 5-HT and 3-HAA were as potent radical trapping antioxidants to eliminate lipid peroxidation, thereby suppressing ferroptotic cell death. 70During this process, monoamine oxidase A markedly abrogated the protective effect of 5-HT via degrading 5-HT.Kynureninase, which was essential for 3-HAA production to confer cells resistant to ferroptotic cell death, whereas 3-hydroxyanthranilate 3,4dioxygenase significantly blocked 3-HAA mediated ferroptosis inhibition. 703-IAA, a Trp metabolite can induce a downregulation of the ROS-degrading enzymes glutathione peroxidase 3 and glutathione peroxidase 7 to reduce tumor proliferation. 71Probiotic L. reuteri promotes interferon-gproducing CD8 T cells, thereby bolstering immune checkpoint inhibitor (ICI) via its released dietary Trp catabolite I3A. 72hers.TMAO engaged in a number of genetic pathways has an apparent association to carcinomas, particularly colon cancer.Mechanistically, TMAO can potentially cause CRC by inflammation, DNA damage, oxidative stress, and protein misfolding. 73However, it remains uncertain whether elevated TMAO levels are a reason or a result of cancer.The level of H 2 S in the feces of CRC subjects is higher than in the control group without tumors. 74H 2 S promotes the proliferation of CRCs via promoting the phosphorylation of Please add expansions for protein kinase B, secondary lymphoid tissue chemokine, CD, inflammatory bowal disease, TGR, HT, Kirsten rat sarcoma viral oncogene homolog, extracellular regulated protein kinase, interluekine, G protein-coupled bile acid receptor 1 (GPBAR1), GATA, PD-L, CRCS and extracellular regulated protein kinase in vitro.In addition, microbiota-derived genotoxin tilimycin also caused colonic stem cell mutations.Urea cycle activation triggered by host-microbiota maladaptation could drive colorectal tumorigenesis.Dietary iron also modulated gut microbiota and induced secretory leukocyte protese inhibitor secretion to promote colorectal tumorigenesis.

Effects of GMMs Associated Immune Cells on the CRCs
GMMs play a critical role in maintaining homeostasis of gut and systemic immunity; however, they also promote immune responses against CRCs (Figure 2).Within CRC microenvironment, there are multiple antitumor immune cells, including multiple kinds of immune cells such as effective CD8 cells, CD4 Th1 cells, Th17 cells, natural killer (NK) cells, dendritic cells (DCs) and inflammatory Macs; whereas other immune cells such as MDSCs, immunosuppressive Macs (M2-like Macs) and N2 neutrophils, Tregs and Bregs can contribute to tumor growth.These tumorassociated immune cells can be regulated by GMMs.Notably, these GMM-mediated immune cells also produce effects on the tumor environments such as that Treg cells in tumor environment can produce vascular endothelial growth factor (VEGF) to promote angiogenesis, and inhibit T effective (Teff) cells, NK cells and antigen-presenting cells by inhibitory cytokines such as IL-10 and cytotoxic molecules such as granzymes and perforin, which can directly kill Teff cells and antigen-presenting cells. 75

Suppression on Immunity Against CRCs by GMMs
SCFAs.SCFAs have an important role in maintaining immune homeostasis through their receptors such as Gprotein coupled receptor (GPR)43.Nucleotide oligomerization domain-like receptor thermal protein domain associated protein 3 (nucleotide oligomerization domain-like receptor 3 (NLRP3))-mediated inflammatory signaling pathways could be negatively regulated by SCFAs to inhibit the activation of Macs.SCFAs also triggered autophagy in cancer cells to promote M2 polarization in Macs, accelerating tumor advancement.7][78] SCFA butyrate also suppressed Th17-associated retinoid-related orphan receptor gt (RORgt) and increased the expression of Treg-associated Foxp3. 79DCs exposed to butyrate facilitated the differentiation of naïve T cells into Foxp3 þ Tregs. 80Administration of SCFAs also increased the Bregs (B regulatory cells) frequency. 81In addition, gut microbiota SCFAs also affected immune effective cells such as that butyrate could decrease the proliferation and cytokine production in Th1, Th17, and Th22 cells. 82 metabolites.BA metabolites have widely effects on immune cells through receptors such as TGR5 (GPBAR1), farnesol-X-receptor, vitamin D receptor (VDR), liver X receptor, pregnane X receptor (PXR), and RORgt.They are essential to maintain tolerant phenotypes of the Macs via TGR5 (GPBAR1).Secondary BAs DCA and LCA could act as endogenous inhibitors of NLRP3 activation by activating TGR5 83 or TGR5-cAMP(adenosine monophosphate)dependent ubiquitination of NLRP3 84 farnesol-X-receptor, another BA receptor, is an important negative regulator of NLRP3. 84BA metabolites could also disrupt intracellular calcium homeostasis, which is essential for nuclear factor of activated T cells signaling and T cells activation. 8524-NorUDCA, a BA metabolite, reshaped immunometabolism in CD8 þ T cells, 86 which affected lympho-blastogenesis, expansion, glycolysis and target of rapamycin complex 1 (mTORC1) signaling.Notably, the physiological concentrations of unconjugated LCA could inhibit the activation of primary human and mouse CD4 þ Th1 cells through a VDRdependent mechanism, resulting in decreased tumor necrosis factor-a and IFN-g. 87VDR activation also promoted a shift from Th1 to Th2 phenotype through increased production of the transcription factors c-Maf and GATA-3. 88ncreased number of tumor-infiltrating Treg cells is associated with poor prognosis in various cancer types. 89Notably, BA metabolites such as 3-oxoLCA, isoalloLCA, and isoDCA have been identified as the regulators of Treg cells by inhibiting the differentiation of Th17 cells and increasing the differentiation of Treg cells in colon.NR4A1 (Nuclear receptor subfamily 4 group A member 1) was also required for the role of isoalloLCA in Treg cells. 90IsoalloLCA could increase binding of NR4A1 at the Foxp3 locus to enhance Foxp3 gene transcription.The differentiation of Th17 cells was also inhibited by 3-oxoLCA by RORgt, 91 which finally affected the Th17/Treg balance.The sulfate of LCA (LCA-3-S) selectively suppressed Th17 cell differentiation without influence on Th1, Th2, and Treg cells. 92Taurohyodeoxycholic acid, a natural 6a-hydroxylated BA, not only reduced the secretion of Th1/Th17-related cytokines and transcription factors, but also increased the production of Th2/ Treg-related cytokines and the expressions of transcription factors in the colon. 93In addition, the BA derivative TDCA also increased MDSCs in the spleen of septic mice. 94MDSCs, as immunosuppressive cells antagonized the activities of T cells by depleting amino acids, and expressing transforming growth factor beta (TGFb) and PD-L1. 95Notably, intratumoral enrichment of F. nucleatum in patients with CRC was associated with enrichment of MDSCs. 96Taurodeoxycholic acid (TDCA) also increased the number of MDSCs in the spleen of septic mice. 94p metabolites.Trp metabolites have an important role in the differentiation and function of immune regulatory cells through their receptors such as ary hydrocarbon receptor (AHR).Administration of AHR ligand indole-3carbinol, a Trp metabolite, was sufficient to restore Treg compartment. 97Notably, in vitro experiments also verified that Trp metabolites IPA inhibited the differentiation of Th17 cells and promoted the differentiation of Treg cells. 98otably, during T regulatory 1 (Tr1) cell differentiation, AHR could be physically associated with c-Maf to activate immunosuppressive cytokines IL-10 and IL-21 promoters. 99,100Differentiation and function of IL-10producing CD19 þ CD21 hi CD24 hi Bregs were also regulated by AHR. 101Trp metabolite IAA from gut microbiota together with LPS could activate transcription factor PXR and nuclear factor-kB to induce the generation of IL-35 þ Breg cells. 1024][105][106][107][108] 3-HAA, a downstream metabolite of Kyn promoted the generation of Foxp3 þ Treg cells and immunosuppressive TGF-b in a nuclear coactivator 7 (NCOA7)dependent pathway. 109Microbiota-derived Trp catabolites mediated the chemopreventive effects of statins on CRC by inhibiting Th 17 cell differentiation by targeting the nuclear receptor, retinoic acid receptor-related orphan receptor gt (RORgt). 110Notably, Trp metabolites could also activate AHR in tumor-associated Macs to suppress antitumor immunity 111 such as that the metabolites of dietary Trp generated by the gut microbiota activated the AHR in myeloid cells, promoting an immune suppressive tumor microenvironment. 112In addition, Trp metabolites were also involved in the regulation of immune effective cells.programmed cell death protein-1 (PD-1), an immunosuppressive molecule in CD8 þ T cells could be upregulated through AHR. 1133-HAA metabolite of Kyn could induce apoptosis of T-cells. 114L-kyn metabolites also caused NK cell death via ROS pathway. 115hers.Gut microbiota urolithin A alleviated colitis in mice by improving gut microbiota dysbiosis, modulating microbial Trp metabolism, and triggering AHR activation. 116rea from Bifidobacterium, could enter into Macs to inhibit the binding efficiency of p-signal transucer and activator of transcription 1 to promotor region, and further skew Macs toward a protumor phenotype characterized by the accumulation of polyamines. 117

Promotion on Immunity Against CRCs by GMMs
SCFAs.There are contradictions about the effects of SCFAs on the immune cells.Besides, SCFAs play an important role in maintaining homeostasis, SCFAs also increase tumorkilling CD8 þ T cells and reduce immune-suppressing Tregs in tumor tissues such as that the supplements using SCFAs or SCFA-producing bacteria increase intratumor T cells and raise the concentration of cytokines interferon-g and tumor necrosis factor-a to result in inhibition of tumor growth. 118ecreased abundance of SCFA-producing taxa such as Coprococcus was subsequently associated with a lower number of intratumoral CD4 þ and CD8 þ cells and peripheral CD4 þ T cells.He et al demonstrated that butyrate could promote the antitumor activity of intratumoral and draining lymph node CD8 þ T cells in an IL-12 signaling pathway-dependent manner in a mouse model. 119Thus, SCFAs have beneficial effects in CRCs.
BA metabolites.BA metabolite CDCA suppressed M2 Mac polarization. 120Mechanistically, it could cause mitochondrial morphology damage, including swelling and reduction of cristae, decreased mitochondrial membrane potential, and elevated mitochondrial calcium level, which resulted in the production of ROS.
Trp metabolites.L. plantarum-derived indole-3lactic acid, a Trp metabolite, could ameliorate colorectal tumorigenesis via epigenetic regulation of CD8 þ T cell immunity.Interventions with L. reuteri or its metabolite indole-3-lactic acid could complement chemoprevention strategies for CRCs. 110Kynurenic acid also modulated the recruitment and aggregation of GPR35-positive Macs to cause a robust Th17 immune response.
Others.Both A. muciniphila and L. rhamnosus activated stimulator of interferon genes-interferon pathways to slow tumor progression.Bifidobacterium, another kind of bacteria, altered the functional capacity of DCs to induce CD8 þ T cell proliferation and IFNg production. 121,122It also promoted Th1 transcriptional differentiation and antitumor immune responses to improve immunocheckpoint blockage efficacy. 40Bacteroides fragilis induced Mac polarization to M1 and upregulated CD80 and CD86 expression on the cells, which could promote innate immunity. 123Enterococcus hirae was able to induce the polarization of immune cells in secondary lymphoid organs towards a Th1 IFNg phenotype, leading to increased ratios of cytotoxic T cells to Tregs in mouse models.Eleven strains combined with immunocheckpoint blockages robustly induced IFN g þ CD8 þ T cells to inhibit tumor growth.Faecalibacterium increased CD4 þ T cell proportion and also reduced Treg cell proportion in peripheral blood.L. plantarum effectively increased expression of the natural cytotoxic receptors, and promoted NK cell activation to trigger innate immunity.

Potential Effects of GMM-Mediated Tumor-Associated Macs (TAMs) and MDSCs on CRC Metastasis
There are many factors that regulate the dissemination and distant organ colonization of CRC cells, including genetic mutations, metastasis-initiating cells, epithelialmesenchymal transition (EMT), and the tumor microenvironment.The GMMs induced immunosuppressive cells such as TAMs and MDSCs can help direct metastatic dissemination by creating a niche which allow tumor colonization.These immunosuppressive cells can achieve protumor functions by generating a proinflammatory cytokines, maintaining an immunosuppressive microenvironment, remodeling the matrix and creating a proangiogenic and proinvasive environment, and secreting growth factors (Figure 3).
For example, GMM-mediated immunosuppressive Macs could release nitric oxide and reactive oxygen intermediates to cause genetic instability during the initiation phase.These Macs also produced epidermal growth factor and mediators such as IL-6, hepatocyte growth factor, and glycoprotein neuromedin B to promote cancer stem cell expansion.These Macs also potentially contributed to metastatic spread by releasing IL-1 and TGFb, which could be involved in extracellular matrix (ECM) remodel and pathological fibrosis at later stages.In addition, these Macs also are a critical source of angiogenic factors such as VEGF and proangiogenic chemokines.
Gut microbiota associated MDSCs are also indispensable for the formation of pre-metastatic niche, which promote the niche to favor tumor cell colonization and promote metastasis. 124Indeed, Peptostreptococcus anaerobius induced chronic inflammation and modulated tumor microenvironment by recruiting MDSCs, TANs (Tumor associated neutrophils) and TAMs. 125F. nucleatum also caused liver metastasis by modulating liver microenvironment with accumulation of MDSCs, and reduction of NK and Th17 cells.Thus, host microbiota metabolites mediated immunosuppressive cells could promote CRC metastasis.Recent studies have shown that tumor-associated neutrophils (TANs), which can be polarized to antitumorigenic N1 and protumorigenic N2 phenotype, can form web-like structures known as neutrophil extracellular traps (NETs) both in primary tumor microenvironment and metastatic sites.Interestingly, NETs were involved in cancer progression and metastasis. 126N2 neutrophils support edtumor growth by expressing arginase, metalloproteinase 9 (MMP-9), VEGF, CCL2, CCL5 and CXCL4. 126

Direct Roles of GMMs in CRC Metastasis
Tumor metastatic formation from primary tumor to metastatic tumor would pass several stages, including hybrid intermediate cells, transient fetal/regenerative progenitors, differentiated stem cell-like MIC (metastasis initiating cell), dormant MIC and proliferative MIC.Studies have found that some bacteria are involved in the induction of metastasis in primary CRC.They include Fusobacterium nucleatum, Enterococcus faecalis, Bacteroides fragilis, E. coli and Salmonella enterica.Pathogen E. coli upregulated cathepsin K (CTSK) expression which served as a vital mediator between the imbalance of intestinal microbiota and CRC metastasis. 127ut microbiota-stimulated cathepsin K could mediate Toll-like receptor 4 (TLR4)-dependent M2 Mac polarization and promote tumor metastasis in CRC. 127Some gut microbiota derived metabolites such as LPS has been found to promote EMT through the upregulation of TGFb-1 and the activation of nuclear factor-kB. 128Clostridium butyricum inhibited EMT of intestinal carcinogenesis through downregulating methyltransferase. 129In addition, cadaverine could inhibit EMT in breast cancer cell lines through modulating the expression of MMP-9.However, studies have shown that SCFAs, BA, and Trp metabolites from gut microbiota also directly affect the shaping of tumor metastasis (Figure 4).
SCFAs.SCFA butyrate impeded the angiogenesis, metastasis, and survival of CRC cells by inhibiting Sp1 transactivation via the neuropilin-1/VEGF pathway. 47It also reduced clone formation, and migration through the extracellular regulated protein kinase2/mitogen-activated protein kinase pathway.Importantly, butyrate could stimulate the proliferation of normal colonocytes and cancerous colonocytes when the Warburg effect was prevented from occurring. 47However, SCFA acetate could promote metastasis by increasing zinc finger protein snail family transcriptional repressor 1 and acyl-CoA synthetase short chain family member 2 in renal carcinoma cells under glucose limitation. 130A metabolites.BAs not only participate in the metastatic colonization but also angiogenesis (Figure 4).Differential BA signals emitted by various BA profiles exert distinct pathophysiological traits to the occurrence and development of CRCs.The conjugated BAs promoted CRCassociated liver metastasis.BA metabolite DCA induced EMT and activated VEGF receptor 2. 131 DCA, as a transcriptional activator in cancer was associated with the enzyme cyclooxygenase 2 in fibroblasts, 132 which had an influence on tumor microenvironment by increasing the invasiveness and proliferation of cancerous cells.In addition, LCA also contributed to cancer progression and metastasis through inducing the expression of urokinasetype plasminogen activator receptor, 133 and increasing the expression of matrix MMP genes, including MMP-1, MMP-2, and MMP-7. 134Meanwhile, LCA could promote IL-8 expression to stimulate CRC angiogenesis.In addition, taurousodeoxycholic acid, a conjugated form of UDCA, had anti-invasive impacts through decreased expression of MMP-7 and MMP-13 in metastatic breast cancer. 135UDCA also decreased the invasiveness of gastric carcinoma cells by interfering with the generation of prostaglandin E2.
Trp metabolites.AHR was involved in cancer initiation and metastasis.Trp metabolites, as AHR ligands, participated in the occurrence and development of cancer. 37deed, D-kyn could promotes epithelial-to-mesenchymal transition via activating AHR. 136PXR, another receptor of Trp metabolites, also affected cancer growth, progression, and chemoresistance by regulating the expression of genes implicated in proliferation, metastasis, apoptosis, inflammation, and oxidative stress.Notably, cytoguardin, a Trp metabolite, could resist against cancer growth and metastasis. 1378-hydroxyquinaldic acid, another Trp metabolite, also exerted antiproliferative and antimigratory effects on CRCs. 138

Gut Microbiota Elicits CRC-Promoting Inflammation
Inflammation is a risk factor and also a hall marker of CRCs.Colonization with the bacteria such as F. nucleatum and B. fragilis was associated with colonic inflammation, which was the principal mechanism in promoting colorectal tumorigenesis. 63,139F. nucleatum could drive a proinflammatory intestinal microenvironment through metabolite receptor-dependent modulation of IL-17 expression in Apc min/þ mice. 140The FadA adhesin/invasin conserved in F. nucleatum is a key virulence factor.B. fragilis could cause a series of inflammatory reactions due to B. fragilis toxin.Others such as TLR2 and TLR4 (TLR2/4) also sense extracellular microbial products, lipopolysaccharide or lipoteichoic acids from Gram-negative and Gram-positive bacteria, activate TLR2/4 signaling to cause downstream expression of proinflammatory cytokines.The nonspecific immunity activation by peptidoglycan involves immunomodulation by different pattern recognition receptors including peptidoglycan recognition proteins, NLRs, TLRs, and C-type lectin receptors.The inflammasomes such as NLRP3 (a central adenosine triphosphatase (ATPase) domain-leucine-rich repeat-pyrin domain-containing proteins 3) also sense pathogen-associated molecular pattern molecules and damage-associated molecular patterns to leads to secretion of IL-1b and IL-18.Recent studies have shown that SCFAs, BA, and Trp metabolites also promote inflammation through immune cells.Indeed, although it has been widely reported that SCFAs play anti-inflammatory roles through different signaling pathways, SCFAs also play proinflammatory roles through G-protein-coupled receptors.For example, Kim et al 141 found that SCFAs activated GPR41 and GPR43 on intestinal epithelial cells to protect immunity and tissue inflammation in C57BL6 mice SCFA acetate promotes this process through SNAl1, whereas butyrate inhibits this process through Warburg effect; BA metabolites CDCA, UDCA, TUDCA, and LCA can promote metastasis of tumor through upregulating COX-2, PGE2, MMP7/13 and uPAR respectively.Trp metabolites also promote metastasis of tumor through PXR and AHR, whereas metastasis can be inhibited by cytoguardin through downregulating E-cad and upregulating N-cad and Vim.8-hydroxyquinaldic acid also inhibits metastasis through downregulating b-cat and E-cad.Other metabolites such as that LPS promotes metastasis through upregulating TGFb1; Whereas CAD can inhibit metastasis through downregulating MMP9.Angiogenesis play an important role in tumor metastatic formation.SCFAs can inhibit the angiogenesis by downregulating VEGF and HIF1a.Notably, angiogenesis can be promoted by BA metabolites CDCA and LCA through upregulating HIF1a and IL-8 respectively, whereas DCA-heparin conjugate inhibits angiogenesis through upregulating bFGF.LPS also promotes angiogenesis by upregulating VEGF.b-FGF, b fibroblast growth factor; AHR, ary hydrocarbon receptor; BA, bile acid; CAD, cadaverine; COX-2, cyclooxygenase-2; CSC, cancer stem cell; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; E-cad, E-cadherin; GMM, gut microbiota metabolite; HIF1a, hypoxia-inducible factor 1a; LCA, lithocholic acid; LPS, lipopolysaccharide; MIC, metastasis initiating cell; MMP, matrix metalloproteinase; N-cad, N-cadherin; PGE2, prostaglandin E2; PXR, pregnane X receptor; SNAI1, snail family transcriptional repressor 1; SCFAs, short chain fatty acids; TGFb, transforming growth factor b; Trp., tryptophan; b-cat,b-catenin; TUDCA, taurousodeoxycholic acid; UDCA, ursodeoxycholic acid; uPAR, urokinase-type plasminogen activator receptor; VEGF, vascular endothelial growth factor; Vim, vimentin.
without free fatty acid receptor 2 or 3 receptors.The impact of BAs on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1b.These studies showed that secondary BAs exerted anti-inflammatory effects, but sulfation of secondary BAs abolished their antiinflammatory properties. 142Thus, gut microbiota-elicited inflammation, at least in part, contributes to both the occurrence and development of CRCs.

Modulation of anti-CRC Immunity by Gut Microbiota
Tumor immunotherapies such as ICIs are effective strategies against tumor.The interactions between gut microbiota and cancer immune responses cause the possibility that gut microbiota can affect tumor immunotherapy.Although there are very few reports on gut microbiota-based methods to enhance immunotherapy efficacy in patients with CRCs, specific bacterial species associated with immunotherapy responses in animal models such as Bifidobacterium spp have been found. 143herapeutic methods that target microbiota are being explored, such as fecal microbiota transplantation and probiotics (individual probiotics or cocktails).Studies have found that some species are beneficial responses to immunotherapy.Administration of particular probiotic strains (for instance Bifidobacterium) has been revealed to improve the efficiency of immunotherapy via enhancement of PD-1/PD-L1 or cytotoxic T-lymphocyte antigen-4 blockade. 144Mager et al 40 revealed that Bifidobacterium pseudolongum, Lactobacillus johnsonii, and Olsenella species were able to significantly improve the efficacy of ICIs in mouse models of cancer via the production of the metabolite inosine. 40B. longum KACC 91563 strain was able to modulate the hosts' immune system via immunoglobulin production as well as acting via the maintenance and improvement in the Th1/Th2 balance. 145Routy et al 146 observed that the abundance of Akkermansia muciniphila positively affected the clinical responses to ICIs.In addition, prebiotics such as the administration of soluble fibre including inulin and pectin could improve the activity of anti-PD-1 antibodies in various mouse model. 147Engineered probiotics and phage-targeted depletion of pathogenic bacteria. 147,148have also been used in therapy against CRC.A tumor-colonizing form of E. coli has been engineered to synthesize arginine from ammonia, thus boosting intratumoral L-arginine availability, which is essential for the proper function of cytotoxic T cells. 149owever, for the development and clinical application of these methods, it is critical to decipher the specialized roles of gut microbiota in regulating the immune responses in CRCs.The findings will give new opportunities to take advantage of our knowledge on the gut microbiota to prevent cancer, augment therapies and reduce adverse effects of treatment.

Conclusion
We here summarize the effects of GMMs, especially SCFAs, BA, and Trp metabolites on the CRCs and CRC-associated immune cells.SCFAs can inhibit the proliferation of CRC cells, but some of SCFAs also improve immune tolerances to promote development of CRCs.BA metabolites possess carcinogenic nature but some secondary BAs also inhibit the proliferation of CRCs, and importantly induce immune tolerance.Trp metabolites can inhibit CRCs through regulating immune cells.For example, dietary fortification with tributyrin, a butyrate glycerol ester, was shown to arrest the development of CRC in mice, implying that butyrate was an effective anticancer metabolite.UDCA, a secondary BA, produced by Ruminococcus gnavus could protect against the development of CRC.However, some of Trp metabolites also directly and indirectly promote the growth of CRCs.Thus, gut microbiota is not only a friend but also a foe of CRCs.
Gut microbiota also directly and indirectly (via immunosuppressive cells) affect tumor metastasis via regulating the shaping of immune-privileged metastatic niches.Since precise and/or personalized treatments of CRCs, which are focused on the gut microbiota, are likely to target either the key cancer-promoting pathogens or the bioactive components or metabolites, all of the findings will increase more opportunities for clinical applications of gut microbiota.